Spontaneous healing of human amnion in the premature rupture of membrane model

Introduction: This study aimed to explore the spontaneous healing of ruptured fetal membranes experimentally in the prelabor rupture of membrane model using the amnion pore culture technique. Methods: The human amniotic membrane was separated from the post-delivery term placenta in women with normal pregnancies who delivered by scheduled unlabored cesarean section and stained immunohistochemically with primary antibodies against SSEA-4, OCT-3/4, and TRA-1-60. The characteristics of the cultured amniotic epithelial cells were examined by fluorescence-activated cell sorting analysis. Amniotic membranes with perforations that were 1, 2, and 3 mm in diameter were cultured in αMEM containing 10% heat-inactivated FBS, 1% penicillin-streptomycin, and 10 ng/mL EGF at 37 °C in a humidified incubator with 5% CO₂. Next, the pore sizes were calculated to evaluate the healing process. Results: The amniotic membrane stained positive for CD49d and pluripotent stem cell markers such as SSEA-4, TRA 1-60, and OCT-4 in the stromal and epithelial cell layers. In the flow cytometry analyses, the extracted amniotic epithelial stem cells were observed to express indicator markers for stem cells such as SSEA-4, OCT-4, SOX-2, and Nanog. In the amnion pore culture technique model, the 1-mm pores healed completely, whereas the 2- and 3-mm pores did not heal substantially. Discussion: The amnion pore culture technique was useful for demonstrating the natural healing process of the human amniotic membrane. Stem cells in the human amnion might facilitate the resealing of small pores in the amniotic membrane, as observed in this model.